TDP1-independent pathways in the process and repair of TOP1-induced DNA damage

Anticancer drugs, such as camptothecin (CPT), trap topoisomerase I (TOP1) on DNA and form TOP1 cleavage complexes (TOP1cc). Alternative repair pathways have been suggested in the repair of TOP1cc. However, how these pathways work with TDP1, a key repair enzyme that specifically hydrolyze the covalent bond between TOP1 catalytic tyrosine and the 3’-end of DNA and contribute to the repair of TOP1cc is poorly understood. Here, using unbiased whole-genome CRISPR screens and generation of co-deficient cells with TDP1 and other genes, we demonstrate that MUS81 is an important factor that mediates the generation of excess double-strand breaks (DSBs) in TDP1 KO cells. APEX1/2 are synthetic lethal with TDP1. However, deficiency of APEX1/2 does not reduce DSB formation in TDP1 KO cells. Together, our data suggest that TOP1cc can be either resolved directly by TDP1 or be converted into DSBs and repaired further by the Homologous Recombination (HR) pathway.

: TDP1-KO cells showed cellular sensitivity to TOP1 poison camptothecin (CPT). a Colony formation assay of HEK293A-WT and TDP1-KO cells with ETO treatment. Data are represented as mean ± SD. n=6 biologically independent replicates. Two-tailed unpaired t test with Welch's correction was used for statistical analysis. ns, not significant. b Colony formation assay of HeLa-WT and HeLa TDP1-KO cells with ETO treatment. Data are represented as mean ± SD. n = 6 biologically independent replicates. Two-tailed unpaired t test with Welch's correction was used for statistical analysis. *p = 0.0179, and ns, not significant. c The proliferation of HEK293A-WT, TDP1-KO-1, and TDP1-KO-4 cells was measured using a CellTiter-Glo assay after 3 days in the presence of the indicated concentrations of CPT. Data are presented as the mean ± SD (n = 3 biologically independent replicates). Two-tailed unpaired t-test was used for statistical analysis of the IC50 of each cell line. ***p (HEK293A-WT vs. TDP1-KO-1) = 0.00029, and ***p (HEK293A-WT vs. TDP1-KO-4) = 0.00029. d as in c but treated with different concentrations of ETO. Data are presented as the mean ± SD (n = 3 biologically independent replicates). Two-tailed unpaired t-test was used for statistical analysis. *p (HEK293A-WT vs. TDP1-KO-1) = 0.01418, and **p (HEK293A-WT vs. TDP1-KO-4) = 0.00363. e as in c but the proliferation of HeLa and HeLa TDP1-KO cells was measured. Data are presented as the mean ± SD (n = 3 biologically independent replicates). Two-tailed unpaired t-test was used for statistical analysis. ***p = 0.00031. f as in c but the proliferation of HeLa and HeLa TDP1-KO cells with ETO treatment was measured. Data are presented as the mean ± SD (n = 3 biologically independent replicates). Two-tailed unpaired t-test was used for statistical analysis. ns, not significant. g WT and TDP1-KO cells were treated with 10 µM CPT for 1 h and then 4 released into fresh medium and cultured for the indicated times. Whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies.
Experiments were repeated at least three times, and similar results were obtained.

Supplementary Fig. 2: TDP1-KO cells show increased DSB-induced DNA damage response after treatment with TOP1 poison in HeLa cells. a HeLa WT and HeLa
TDP1-KO cells were treated with 10 µM CPT for the indicated times. Whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies.
Experiments were repeated at least three times, and similar results were obtained. b HeLa WT and HeLa TDP1-KO cells were pre-treated with 10 µM ATRi (AZD6738), 1 µM ATMi (AZD0156), 10 µM DNA-PKi (AZD7648), or 10 µM MG132 for 1 h and then independent experiments is shown in a bar chart (mean ± SD, n = 3 biologically independent experiments). A two-tailed unpaired t test with Welch's correction was used for statistical analysis. ns, not significant. e Percentage of pDNA-PKcs-S2056-positive cells from three independent experiments was shown in a bar chart (mean ± SD, n = 3 biologically independent experiments). A two-tailed unpaired t test with Welch's correction was used for statistical analysis. f A RADAR assay was performed after treating the indicated cells with 10 μM CPT for 1 h to assess TOP1cc accumulation.
Data are represented as mean ± SEM (n = 4 biologically independent experiments). A two-tailed unpaired t test with Welch's correction was used for statistical analysis. ns, not significant.